Troponin is a regulatory protein for contraction of the striated muscle, present in myocardium and skeletal muscle, and consists of three subunits, i.e., TnI, TnC, and TnT. The three subunits form a complex that plays an important regulatory role in muscle contraction and relaxation. TnI is an inhibitory subunit of actin, which has three subtypes: fast skeletal subtype (fTnI), slow skeletal muscle subtype (sTnI), and cardiac subtype (cardiac troponin I, cTnI). The cTnI has a molecular mass of 23.9 kD, with 210 amino acid residues. Increased levels of cTnI may be an important serological marker for diagnosis of acute myocardial infarction (AMI). When acute myocardial infarction occurs, cTnI is released into the bloodstream within 4-8 hours due to myocardial obstruction, so that its concentration is beyond the concentration range for a healthy person. In general, cTnI concentrations are highest in 12-18 hours following onset of AMI and remain for 5 to 10 days.
Acute myocardial infarction is myocardial necrosis caused by acute and persistent ischemia and hypoxia of coronary artery. Severe and persistent posterior sternal pain is common clinically, which cannot be completely relieved by rest and nitrate drugs, and accompanied by increased levels of serum myocardial enzymes and progressive ECG changes, which can be further complicated by arrhythmia, shock, or heart failure, and even life-threatening. It is one of the common cardiology diseases, which seriously endangers human health. This disease is most common in Europe and the United States. In the United States each year about 1.5 million people have an onset of myocardial infarction. In recent years, China showed a clear upward trend, with the number of new cases every year being at least 0.5 million and at least 2 million patients currently suffering. In recent years, great progress has been made in treating AMI, changing from passive conservative treatment to positive elimination of thrombosis or percutaneous coronary artery dilatation, and even coronary artery bypass surgery. This set a high standard for early accurate diagnosis of AMI, while the AMI attack sensitivity is only about 50%. The sensitivity gradually increases with time to be 90% or more at 6 h, showing a tendency of increasing sensitivity over time, which means that the key issue in early accurate diagnosis of AMI is to increase the sensitivity of the assay method.
At present, AMI assay methods mainly include characteristic ECG changes and dynamic changes of serum biomarker. However, about 25% of myocardial infarction patients have no typical clinical symptoms during the early stage of the disease, about 50% of AMI patients lack ECG-specific changes. With ECG changes and clinical symptoms alone, AMI diagnostic accuracy was merely 75%. In this case, the detection of myocardial injury markers is particularly important in the diagnosis of AMI. Myocardial injury markers are mainly creatine kinase isoenzyme (CK-MB) and cTnI. Since CTnI has the advantages of rapid dynamic release, complete curve, distinct peak, strong tissue specificity, long diagnosis window period, rapid assay process, and early appearing in blood after myocardial injury, the AMI diagnostic method based on cTnI is preferred over the AMI diagnostic method based on CK-MB, and has been widely accepted clinically. CTnI has not only become the “gold standard” for diagnosis of acute myocardial infarction, but also has become the most suitable marker for monitoring, clinical observation, risk classification, and prognosis evaluation of myocardial disease.
There are many ways of cTnI determination, mainly including radioisotope immunoassay (RIA), enzyme-linked immunosorbant assay (ELISA), and colloidal gold immunochromatography (ICA), and so on. In recent years, with the development of cTnI research, some new and more accurate assay methods were developed in the clinical laboratory, such as chemiluminescence (CLIA).
Radioisotope immunoassay has many problems, such as complicated operation, long testing time, unsuitablility for large-batch assay, poor reproducibility, and radionuclide contamination.
The mechanism for Enzyme-linked immunosorbant assay is using a specific antibody coated with a solid-phase carrier as the first antibody, adding serum to be tested, then add a biotinylated second antibody to form a double sandwich, washing for separation after incubation, and adding a luminescent matrix substrate. The concentration of cTnI was obtained by comparing the measured luminous intensity of the matrix with the standard curve. However, the enzyme-linked immunosorbant assay has many disadvantages such as complicated operation, long measurement period, relatively low sensitivity, narrow linear range, and especially the low content of serum cTnI, the detection of cTnL by ELISA method showed obviously poor sensitivity, which makes it impossible to quickly diagnose the onset of acute myocardial infarction (AMI) in the early stage, limiting further application of ELISA in the clinical detection of cTnI.
Colloidal gold immunochromatography uses less sample and is simple and fast, suitable for cTnI bedside detection. The basic mechanism is to detect cTnI by use of the binding of two anti-cTnI monoclonal antibodies. When the serum sample is dropped into the absorption hole, a first gold-labeled antibody and cTnI bind to form an antibody-antigen complex, and a second immobilized cTnI monoclonal antibody captures this complex, generating a pink band, while there is no band present in the reaction loop in case of absence of the complex. However, colloidal gold immunochromatography can only apply for qualitative determination and is of poor sensitivity for quantitative determination. Test cards developed based colloidal gold immunochromatography, though capable of rapid qualitative detection of cTnI, also has the problem of the low sensitivity. Early in AMI, when the serum contains only a small amount of cTnI, colloidal gold immunochromatography cannot provide accurate diagnosis of AMI onset, and thus its clinical application is still greatly restricted.
Chemiluminescence (CL) is a type of labeled immunoassay technique for detecting trace antigen or antibody by combining luminescence analysis and immunological reaction, which comprises two parts, i.e., immunological reaction system and chemiluminescence analysis system. Chemiluminescence analysis system uses chemiluminescent substances to form an intermediate of an excited state via catalysis by a catalyst and oxidation by an oxidant. When the excited intermediate goes back to a stable ground state, it will emit photons at the same time; the quantum yield of light is measured with the use of luminescence signal measuring instrument. The immune response system will mark the marker material directly on the antigen or antibody, form antigen—antibody complex by a specific reaction, and then detect by a detection method of the corresponding marker. CLIA's main advantages are its high sensitivity, wide linear range, long marker lifetime, no radioactive hazards, and potential for full automation.
The cTnI kit supplied by Siemens includes a ReadyPack® Master Kit containing the ADVIA Centaur® High Sensitivity TnI™ Double Labeling Reagent, solid phase reagents, and adjuvant reagents. The double labeling reagents consist of acridinine-labeled goat polyclonal anti-cTnI antibody (˜0.15 μg/mL) and two biotin-labeled mouse monoclonal anti-cTnI antibody (˜2.0 μg/mL). The solid phase reagent is a latex magnetic particle suspension. The adjuvant reagent is a non-magnetic latex particle. The ADVIA Centaur Ultrasensitive TnI assay of this manufacture is a 3-point sandwich immunoassay using direct chemiluminescence technique. During the assay, the antibody in the double labeling reagent binds to troponin I in the sample, producing an immune complex. The biotin contained in the immune complexe randomly binds to labeled streptavidin on the magnetic particles.
Roche provides a cTnI assay kit, which can bind with the antigen with biotinylated anti-cTnI monoclonal antibody and ruthenium (Ru) complex-labeled anti-cTnI monoclonal antibody to form a sandwich structure, and detect cTnI by electrochemically luminescence Immunoassay (ECLIA).
Chemiluminescence method has the advantages of accuracy, strong specificity, and good precision, while its sensitivity is much higher than that of enzyme-linked immunosorbant assay and colloidal gold immunochromatography. However, the sensitivity of existing cTnI commercial test kit for clinical applications is still not high enough, with an analytical sensitivity of 5 pg/mL or above, and thus cannot accurately detect ultra-low concentrations of cTnI or meet the high standards of early diagnosis of AMI.